Stem cells are generally defined as clonogenic cells capable of both self-renewal and multi-lineage differentiation. Post-natal stem cells have been isolated from various tissues, including bone marrow, adipose, skin, retina, and extra-embryonic tissues such as placenta, umbilical cord amniotic tissues. Along with this, dental tissue (DT) appears to be an excellent source for stem cells because it can be obtained as part of a planned serial extraction for management of occlusion. The DT can be isolated from various age groups and teeth; for example, cells isolated from dental tissue of human impacted tooth germ are known as tooth germ progenitor cells (TGPCs), stem cells from human exfoliated deciduous teeth are known as SHED, and stem cells can also be isolated from human permanent teeth (impacted molar) (DPSCs) or from apical papilla (SCAP) and periodontal ligament tissues (PDLSCs). DT offers an unlimited source of human mesenchymal stromal/stem cells (MSCs) for cell replacement/regeneration therapy as it has been shown to be multifaceted, ranging from improved recovery from stroke, cardiac ischemia to wounds and burns. However, optimal culture conditions for their long-term expansion and proliferation are necessary since large numbers of stem cells are required for therapeutic/clinical applications.
Once the precursor cells are isolated from dental tissues, maintaining their vitality is of utmost importance and various storage media have been suggested in the prior art that can be used for preserving the vitality of the isolated cells. One of the most widely used medium for storing isolated dental tissues is Eagle's Medium. Eagle's medium was first described in the article by M. Eagle, entitled “Amino acid metabolism in mammalian cell cultures”, in Science, vol. 130, pages 432-437 (1959). Its specific use in preserving Monkey incisors in tissue culture before replantation has been reported by J. O. Andreasen et al, in the International Journal of Oral Surgery, vol. 7, pages 104-112 (1978). Another artificial medium for preserving or storing the isolated dental tissue is the Hanks Balanced Salt Solution.
One of the foremost requirements for the artificial media for storing and preserving the isolated tissue from dental origin is that its composition should resemble with the fluids present in the isolated cells. This kind of resemblance between the composition of the storage media and the cell fluid is essential since it prevents the undue inflow or outflow of ions from the isolated cells thereby helping them maintain their vitality. In this respect, Eagle medium as well as the Hanks solutions has been proved to be effective in maintaining the vitality of the isolated dental tissue for practically significant period of time. Use of such storage media for maintaining the vitality of the periodontal membrane of the ex-articulated teeth has been disclosed in EP 0251291.
PCT Application WO 2011/064733 is directed to the isolation of human mesenchymal cells and their large scale proliferation for the purposes of obtaining a cell composition for the treatment of various diseases, teaches a medium comprising Dulbecco's Modified Eagle's Medium Knock-Out [DMEM-KO], Fetal Bovine Serum (FBS), Glutamine and Pen-Strep for collecting the isolated tissue. Further, WO 2012/117333 which specifically deals with isolation and proliferation of the DPSCs, discloses a transport medium or a preserving medium for ensuring the vitality of the isolated dental tissue before it is further subjected to expansion and proliferation. The transporting medium as disclosed in WO 2012/117333 comprises Dulbecco's Modified Eagle's Medium-Knock Out (DMEM-KO), Fetal Bovine Serum (FBS), Pen-Strep, Glutamine, Ascorbic acid and Insulin-Transferrin-Selenium (ITS).
Having isolated and stored the dental tissue, the next challenge is to amplify or expand the isolated mesenchymal cells. Again various types of media for in vitro expansion of human mesenchymal cells have been disclosed. For example, the present applicant in one of their co-pending PCT applications WO 2008/129563, have disclosed a growth media for expansion and sub-culturing the isolated mesenchymal cells that includes a basal medium comprising KO-DMEM, DMEM-LG and DMEM-F12 along with one or more of other constituents such as fetal bovine serum (FBS), growth factors, 200 niM glutamax, antibiotics, human plasma and heparin. Another PCT Application in the name of the present applicant WO2011/06733 that deals with Mesenchymal stem cells obtained from human bone marrow (hBMSCs), discloses a growth media comprising DMEM-KO supplemented with 10% FBS, 200 mM Glutamax and Pen-Strep. The requirements for the media used for expansion are different for each type of isolated tissue. Further, these requirements also vary depending on the nature of the intended differentiated tissue one intends to develop from the isolated cells. Particularly, in the context of DPSC, the present applicant in another co-pending PCT application WO 2012117333 has disclosed a xeno-free culture medium that comprises of DMEM-KO, Human Platelet Lysate (HPL), penicillin/streptomycin and Glutamine. KR 20080104274 describes a method for isolating from the follicular sack a new non-haematopoietic, mesenchymal stem subpopulation, referred to as FENC (Follicle-derived Embryonic Neural Crest stem cells), wherein variety of different media for each of type of the intended cell differentiation.
The presently known preservation media or expansion media used for isolation and expansion of precursor cells from dental origin suffer from shortcomings. Most of these media contain ascorbic acid. As is well known in the art, ascorbic acid is an extremely sensitive chemical and it is very susceptible to oxidation upon its exposure to oxygen at ambient temperature. Ascorbic acid present in these media therefore can get oxidized within a very short period of time which may even be lesser than 48 hours. The oxidized product can harm the tissues and thereby severely compromising the preservative ability of the medium. Another relatively unstable ingredient in the preservation/expansion media described above is Glutamine. At ambient temperatures, Glutamine upon degradation leads to the formation of ammonia which severely affects the preservation/expansion ability of such media. Another shortcoming of the conventionally known media used for isolation and expansion of stem cells is that they are usually prone to contamination and they are not easy to prepare and they are usually not cost-effective. Given the specific nature of the requirements for preservation and expansion of various precursor cells from dental origin, there still remains a need for a sample collection media and an expansion media that is simple, cost-effective, widely applicable and above all which ensures effective preservation of the vitality of the isolated precursor cells from the dental origin for a longer period of time with minimum chances of contamination.
Apart from the collection media, the starting material of dental tissues for the isolation of stem cells is small and it is impossible to scale up the stem cells from dental tissues at early passage using small scale flasks (T-25 cm2 or T-75 cm2). To overcome this caveat, the present invention introduced the large scale expansion system wherein the cells were cultured gradually from a 25 cm2 area culture flask to 6360 cm2 area culture flask. This resulted in more than 200 million cells than can cater for the needs for both autologous and allogeneic transplantation, considering 70-kg patients need approximately 2×106 cells/kg body weight for transplantation.